Tests show new biosensor can guide environmental clean ups

Money PointThe Money Point site during dredging of PAH-contaminated sediments. The orange boom contains the released sediment. The VIMS scientists operated the biosensor from the small boat near the boom.
Photo courtesy of Joe Rieger, Elizabeth River Project.

Results from the biosensor show a spike in PAH levels during a rainfall event, and then a gradual decline after the rain has stopped.
Graph courtesy of Candace Spier.

Device can also provide an early warning system for spills

Tests of a new antibody-based “biosensor” developed by researchers
at the Virginia Institute of Marine Science show that it can detect marine
pollutants like oil much faster and more cheaply than current technologies. The
device is small and sturdy enough to be used from a boat.

Testing of the biosensor in the Elizabeth River and Yorktown Creek, which both drain into
lower Chesapeake Bay, shows that the instrument can process samples in less than 10 minutes,
detect pollutants at levels as low as just a few parts per billion, and do so
at a cost of just pennies per sample. Current technology requires hours of lab
work, with a per-sample cost of up to $1,000.

“Our biosensor combines the power of the immune system with
the sensitivity of cutting-edge electronics,” says Dr. Mike Unger of VIMS. “It holds
great promise for real-time detection and monitoring of oil spills and other
releases of contaminants into the marine environment.”

The biosensor was developed and tested by Unger, fellow VIMS
professor Steve Kaattari, and their doctoral student Candace Spier, with
assistance from marine scientist George Vadas. The team’s report of field tests
with the sensor appears in this month’s issue of Environmental Toxicology and Chemistry.

The instrument was developed in conjunction with Sapidyne
Instruments, Inc., with funding from the state of Virginia, the Office of Naval
Research, and the Cooperative Institute for Coastal and Estuarine Environmental
Technology, a partnership between NOAA and the University of New Hampshire.

The tests in the Elizabeth River took place during clean up
of a site contaminated by polycyclic aromatic hydrocarbons (PAHs), byproducts
of decades of industrial use of creosote to treat marine pilings. The U.S.
Environmental Protection Agency considers PAHs highly toxic and lists 17 as
suspected carcinogens.

The biosensor allowed the researchers to quantify PAH
concentrations while the Elizabeth River remediation was taking place,
gaining on-site knowledge about water
quality surrounding the remediation site. Spier
says the test was “the first use of an antibody-based biosensor to guide
sampling efforts through near real-time evaluation of environmental
contamination.”

In the Yorktown Creek study, the researchers used the biosensor
to track the runoff of PAHs from roadways and soils during a rainstorm.

Biosensor development

Kaattari says “Our basic idea was to fuse two different
kinds of technologies— monoclonal antibodies and electronic sensors—in order to
detect contaminants.”

Antibodies are proteins produced by the immune system of
humans and other mammals. They are particularly well suited for detecting
contaminants because they have, as Kaattari puts it, an “almost an infinite
power to recognize the 3-dimensional shape of any molecule.”

Mammals produce antibodies that recognize and bind with large
organic molecules such as proteins or with viruses. The VIMS team took this
process one step further, linking proteins to PAHs and other contaminants, then
exposing mice to these paired compounds in a manner very similar to a regular
vaccination.

“Just like you get vaccinated against the flu, we in essence
are vaccinating our mice against contaminants,” says Kaattari. “The mouse’s
lymphatic system then produces antibodies to PAHs, TNT, tributyl tin [TBT, the
active ingredient in anti-fouling paints for boats], or other compounds.”

Once a mouse has produced an antibody to a particular
contaminant, the VIMS team applies standard clinical techniques to produce
“monoclonal antibodies” in sufficiently large quantities for use in a biosensor.

“This technology allows you to immortalize a lymphocyte that
produces only a very specific antibody,” says Kaattari. “You grow the
lymphocytes in culture and can produce large quantities of antibodies within a
couple of weeks. You can preserve the antibody-producing lymphocyte forever, which
means you don’t have to go to a new animal every time you need to produce new
antibodies.”

From antibody to electrical signal

The team’s next step was to develop a sensor that can
recognize when an antibody binds with a contaminant and translate that recognition
into an electrical signal. The Sapidyne® sensor used by the VIMS team works via what Kaattari
calls a “fluorescence-inhibitory, spectroscopic kind of assay.”

In the sensor used on the Elizabeth River and Yorktown Creek,
antibodies designed to recognize a specific class of PAHs were joined with a
dye that glows when exposed to fluorescent light. The intensity of that light
is in turn recorded as a voltage. The sensor also houses tiny plastic beads that
are coated with what Spier calls a “PAH surrogate”—a PAH derivative that
retains the shape that the antibody recognizes as a PAH molecule.

When water samples with low PAH levels are added to the
sensor chamber (which is already flooded with a solution of anti-PAH antibodies),
the antibodies have little to bind with and are thus free to attach to the
surrogate-coated beads, providing a strong fluorescent glow and electric
signal. In water samples with high PAH concentrations, on the other hand, a
large fraction of the antibodies bind with the environmental contaminants. That
leaves fewer to attach to the surrogate-coated beads, which consequently
provides a fainter glow and a weaker electric signal.

During the Elizabeth River study, the biosensor measured PAH
concentrations that ranged from 0.3 to 3.2 parts per billion, with higher PAH levels
closer to the dredge site. In Yorktown Creek, the biosensor showed that PAH
levels in runoff peaked 1 to 2 hours after the rain started, with peak concentration
of 4.4 parts per billion.

Comparison of the biosensor’s field readings with later
readings from a mass spectrometer at VIMS showed that the biosensor is just as
accurate as the more expensive, slower, and laboratory-bound machine.

A valuable field tool

Spier says “Using the biosensor allowed us to quickly survey
an area of almost 900 acres around the Elizabeth River dredge, and to provide information
about the size and intensity of the contaminant plume to engineers monitoring
the dredging from shore. If our results had shown elevated concentrations, they
could have halted dredging and put remedial actions in place.”

Unger adds “measuring data in real-time also allowed us to
guide the collection of large-volume water samples right from the boat. We used
these samples for later analysis of specific PAH compounds in the lab. This
saved time, effort, and money by keeping us from having to analyze samples that
might contain PAHs at levels below our detection limit.”

“Biosensors have their constraints and optimal operating
conditions,” says Kaattari, “but their promise far outweighs any limitations.
The primary advantages of our biosensor are its sensitivity, speed, and
portability. These instruments are sure to have a myriad of uses in future environmental
monitoring and management.”

One promising use of the biosensor is for early detection
and tracking of oil spills. “If biosensors were placed near an oil facility and there was a spill, we would
know immediately,” says Kaattari. “And because we could see concentrations
increasing or decreasing in a certain pattern, we could also monitor the
dispersal over real time.”